Region Capture Micro-C reveals coalescence of enhancers and promoters into nested microcompartments

Goel, Viraat Y.; Huseyin, Miles K.; Hansen, Anders S.

doi:10.1038/s41588-023-01391-1

Cited by 102 publications

(57 citation statements)

References 59 publications

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“…Topologic approaches like RCMC provide critical insight into 3D organization of a locus and may nominate candidate E-P gene interactions. 9,18,20 However, topology alone cannot assess the functional significance of detected contacts. 2,10 Thus, we performed a parallel analysis using flow cytometry-based, tiled CRISPRa screening to identify functional PTEN enhancers.…”

Section: Resultsmentioning

confidence: 99%

“…We performed the protocol as preliminarily described, with some modifications. 18 Briefly, 2.5x10 7 HCT116 cells were crosslinked with DSG and formaldehyde, in biologic duplicate. The chromatin was digested to mononucleosomes utilizing a micrococcal nuclease (MNase) digestion first optimized via titration, followed by end-repair with biotin labeling, and proximity ligation.…”

Section: Methodsmentioning

confidence: 99%

“…Analyses of RCMC and Hi-C datasets were performed as preliminarily described. 18 Briefly, paired-end reads were aligned to the UCSC hg38 genome using bowtie2 (v2.3.5.1), and aligned read mates were subsequently processed with pairtools (v0.3.0) to remove PCR duplicates and non-uniquely mapped reads, index reads, and filter to retain only reads with both mates within the captured region ( Extended Data Table 3 ). 17,51,52 Read counts were binned across the genome for 50bp bins to coarser resolutions using cooler (v0.8.11).…”

Section: Methodsmentioning

confidence: 99%

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IntegrativePTENEnhancer Discovery Reveals a New Model of Enhancer Organization

Cerda-Smith,

Hutchinson,

Liu

et al. 2023

Preprint

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Enhancers possess both structural elements mediating promoter looping and functional elements mediating gene expression. Traditional models of enhancer-mediated gene regulation imply genomic overlap or immediate adjacency of these elements. We test this model by combining densely-tiled CRISPRa screening with nucleosome-resolution Region Capture Micro-C topology analysis. Using this integrated approach, we comprehensively define the cis-regulatory landscape for the tumor suppressor PTEN, identifying and validating 10 distinct enhancers and defining their 3D spatial organization. Unexpectedly, we identify several long-range functional enhancers whose promoter proximity is facilitated by chromatin loop anchors several kilobases away, and demonstrate that accounting for this spatial separation improves the computational prediction of validated enhancers. Thus, we propose a new model of enhancer organization incorporating spatial separation of essential functional and structural components.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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IntegrativePTENEnhancer Discovery Reveals a New Model of Enhancer Organization

Cerda-Smith,

Hutchinson,

Liu

et al. 2023

Preprint

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“…In the recent years, polymer physics has provided concrete frameworks to characterize and rationalize the mechanisms driving the spatiotemporal dynamics of chromosomes: from SMC-mediated loop extrusion for TAD formation , to epigenomic–driven interactions for chromatin 3D compartmentalization. , With recent experimental advances, such as Micro-C , or chromosome tracing, , it may be now possible to study a specific locus at near-nucleosome resolution (200 bp–1 kbp) with good statistics. Hence, more detailed polymer models are required to finely study the organization of specific regions of interest and the interplay between the various physicochemical mechanisms that may act locally …”

Section: Introductionmentioning

confidence: 99%

Topological Constraints and Finite-Size Effects in Quantitative Polymer Models of Chromatin Organization

Abdulla,

Tortora,

Vaillant

et al. 2023

Macromolecules

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Polymer physics simulations have provided a versatile framework to quantitatively explore the complex mechanisms driving chromosome organization. However, simulating whole chromosomes over biologically relevant time scales at a high resolution often constitutes a computationally intensive task, while genes or other regions of biological interest may typically only span a small fraction of the full chromosome length. Conversely, only simulating the subchromosomal region of interest might provide an oversimplistic or even wrong description of the mechanism controlling the 3D organization. In this work, we characterize what should be the minimal length of the chromosome to be simulated in order to correctly capture the properties of a given restricted region. In particular, since the physics of long, topologically constrained polymers may significantly deviate from those of shorter chains, we theoretically investigate how chromosomes being a long polymer quantitatively affects the structure and dynamics of their subsegments. We show that increasing the total polymer length impacts on the topological constraints acting on the system and thus influences the compaction and mobility of subchains. Depending on the entanglement properties of the system, we derive a phenomenological relation defining the minimal total length to account for to maintain a correct topological regime. We finally detail the implications of these conclusions in the case of several biological systems.

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“…In some experiments, in contrast to the global loss of TADs and CTCF loops, in the absence of cohesin (Rao, Huang et al 2017, Schwarzer, Abdennur et al 2017, Wutz, Várnai et al 2017, enhancer-promoter interactions are not completely abolished (Thiecke, Wutz et al 2020, Liu, Maresca et al 2021, Aljahani, Hua et al 2022, Calderon, Weiss et al 2022. In line with these observations, it is of interest that two recent reports based on analysis with Micro-C (Hsieh, Cattoglio et al 2022) and a targeted MNase-based 3C (Goel, Huseyin et al 2023) did not detect clear changes in enhancer-promoter interactions upon cohesin depletion in mES cells. It is possible that these different reports reflect the different degrees to which cohesin may have been depleted, which has previously been observed with similar chromatinbound proteins such as CTCF (Nora, Goloborodko et al 2017, Khoury, Achinger-Kawecka et al 2020.…”

Section: Discussionmentioning

confidence: 56%

Loop extrusion by cohesin plays a key role in enhancer-activated gene expression during differentiation

Stolper,

Tsang,

Georgiades

et al. 2023

Preprint

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Enhancers and their target promoters often come into close physical proximity when activated. This proximity may be explained by a variety of mechanisms; most recently via cohesin-mediated chromatin loop extrusion. Despite this compelling hypothesis, acute depletion of cohesin does not cause widespread changes in gene expression. We have tested the role of cohesin-mediated loop extrusion on gene expression at the mouse alpha-globin locus during erythropoiesis. Acute depletion of cohesin downregulates alpha-globin expression at early but not late stages of differentiation. When single or multiple CTCF sites are placed between the alpha-globin enhancers and promoters, alpha-gene expression is downregulated. Importantly, the orientation of the CTCF site plays a critical role, suggesting that within this activated domain, cohesin predominantly but not exclusively translocates from the enhancers to the promoters. We find that loop extrusion does play an important role in establishing enhancer-promoter proximity and consequent expression of inducible genes during differentiation.

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Region Capture Micro-C reveals coalescence of enhancers and promoters into nested microcompartments

Cited by 102 publications

References 59 publications

IntegrativePTENEnhancer Discovery Reveals a New Model of Enhancer Organization

IntegrativePTENEnhancer Discovery Reveals a New Model of Enhancer Organization

Topological Constraints and Finite-Size Effects in Quantitative Polymer Models of Chromatin Organization

Loop extrusion by cohesin plays a key role in enhancer-activated gene expression during differentiation

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